Impact of inflammation-induced oxidative stress on the integrity of immuno-haematopoietic cells and potential ameliorating interventions in an in vitro HIV model

Wanjiku, Samuel Mburu (2013-12)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Chronic inflammation and immune activation are hallmarks of HIV infection, resulting in chronic oxidative stress with over-utilization of antioxidant defences, which may contribute to the loss of immune cells and faster disease progression. Low levels of antioxidants in HIV- infected individuals have been associated with frequent opportunistic infections and an increased risk of mortality. HIV infection is also associated with on-going and aberrant activation of both the innate and adaptive immune systems. An important aspect of innate immune stimulation is derived from the leakage of lipopolysaccharide (LPS) across the damaged mucosal lining of the gut in early HIV infection. The impact of this innate immune stimulation on the adaptive arm of the immune system, as represented in this study by levels of CD4+ T-cell activation and death, have not been explored previously in untreated HIV infection. Using an integrated approach of immune activation, inflammation, oxidative stress and ameliorating antioxidant intervention for the first time, this thesis reports the impact of inflammatory induced-oxidative stress on CD4+ T-cells in an in vitro HIV model. In a preliminary study, baseline levels of neutrophil respiratory burst as an in vitro indication of immune stimulation were investigated. The relationships between baseline total antioxidant status (TAS), Red blood cell (RBC) antioxidant enzyme activities (catalase, superoxide dismutase & glutathione peroxidase), lipid peroxidation and glutathione redox ratio and other markers of disease in asymptomatic, untreated HIV infection were also explored. The design and optimization of an assay that could determine the effects of LPS-induced oxidative stress on CD4+ T-cells, was a critical part of this study. The development of this assay enabled the measurement of the effects of selected antioxidant interventions N-acetyl cysteine (NAC) and vitamin C, on LPS-induced CD4+ T-cell activation and death. The results were also correlated with CD4 count, viral load and markers of inflammation (fibrinogen & D-dimers) in HIV-infected and uninfected groups. Neutrophils from HIV-infected persons at rest showed a ―primed‖ response to low stimulating agent, bacterial N-formyl peptides (fMLP), which was significantly (P = 0.04) higher than uninfected individuals. There was increased oxidative stress as evidenced by increased catalase activity, malondialdehyde (MDA) and conjugated dienes (CDs) with a corresponding decrease in antioxidant capacity in HIV-infected individuals with lower CD4 count. NAC in combination with vitamin C, significantly (P = 0.0018) reduced activation of CD4+ T-cells to a greater degree than with either antioxidant alone. NAC and vitamin C individually and in combination significantly (P = 0.05, P = 0.012 and P<0.0001) decreased the expression of the markers of apoptosis, Annexin V and 7-amino-actinomycin (7-AAD). Importantly, the antioxidant combination decreased MDA values and significantly (P = 0.01) increased the glutathione redox ratio in the HIV-infected group. Based on these results, the respiratory burst and LPS-induced activation may be important contributing factors in inflammatory-associated oxidative stress in HIV infection and contribute to the depletion of CD4+ T-cells in the asymptomatic stage of HIV infection. These results also indicate the potential inhibitory effects of NAC and vitamin C in combination as agents that may limit immune activation and inflammation-induced oxidative stress. Importantly, the study showed that at this asymptomatic stage, CD4+ T-cells of the HIV-infected group responded similarly to stimulation as the HIV negative group, indicating that antioxidant defences were still functional and that appropriate early intervention at this stage may be protective against oxidative damage to the immune cells. To the best of our knowledge, this study is the first to use an integrated approach involving not only plasma levels of antioxidant status, but also RBC antioxidant enzyme activities, oxidative damage (lipid peroxidation), inflammation, cellular levels of immune activation and potential ameliorating interventions in evaluating the problem of inflammation-induced oxidative stress in HIV infection. Based on the results of this study, it is envisaged that an insight into the immune activation, inflammatory and oxidative stress status of patients will enable long-term profiling of each patient with a view to individualized therapy. This approach may have a direct impact on patient care in resource-limited settings such as sub-Saharan Africa.

AFRIKAANSE OPSOMMING: Chroniese inflammasie en immuunaktivering is kenmerke van MIV-infeksie. Dié twee prosesse lei tot chroniese oksidatiewe stres en oorbenutting van antioksidant verdedigingstelsels, wat lei tot die verlies van die immuun selle en vinniger siektevordering. Lae vlakke van antioksidante in MIV-positiewe individue stem ooreen met gereelde opportunistiese infeksies en 'n verhoogde risiko van mortaliteit. MIV-infeksie word ook geassosieer met langdurige en abnormale aktivering van beide die ingebore en aanpasbare immuunstelsels. 'n Belangrike aspek van ingebore immuun stimulasie in die raamwerk van vroeë MIV-infeksie, is die lekkasie van LPS oor die beskadigde slymvlies voering van die dunderm. Die impak van die ingebore immuun stimulasie op die aanpasbare arm van die immuunstelsel, soos aangetoon in hierdie studie deur die vlakke van CD4 T-sel aktivering en apoptose, is nog nie voorheen ondersoek in onbehandelde MIV-infeksie nie. Met behulp van 'n oorspronklike, geïntegreerde benadering van immuun aktivering, inflammasie, oksidatiewe stres en 'n lae vlak van antioksidant intervensie, lewer hierdie tesis verslag oor 'n in vitro model van inflammasie-geïnduseerde oksidatiewe stres op CD4 T-selle. In 'n voorlopige studie, is basislyn vlakke van die neutrofiel respiratoriese uitbarsting as 'n in vitro aanduiding van immuunstimulasie ondersoek. Die verhoudinge tussen basislyn totale antioksidant status (TAS), rooi bloed sel (RBC) antioksidant ensiemaktiwiteit (katalase, superoksied dismutase, en glutatioon peroksidase), lipied peroksidasie en glutatioon redoks-verhouding, asook ander merkers van siektevordering in asimptomatiese, onbehandelde MIV-infeksie is ook ondersoek. Die ontwerp en optimisering van 'n toets wat die effek van LPS-geïnduseerde oksidatiewe stres op CD4 T-selle kan bepaal, was 'n kritieke deel van hierdie studie. Die ontwikkeling van hierdie toets het ook die meting van die effek van toevoeging van twee geselekteerde anti-oksidante, N-asetiel sisteïen (NAC) en vitamien C, op LPS-geïnduseerde CD4 T-sel aktivering en apoptose ondersoek. Die resultate is ook gekorreleer met CD4-telling, virale lading en merkers van inflammasie (fibrinogeen en D-dimere) in groepe met en sonder MIV-infeksie. Neutrofiele van asimptomatiese persone met MIV infeksie, het 'n 'voorbereide' reaksie gehad teen ‗n lae stimulerende agent, bakteriële N-formiel peptied (fMLP), wat beduidend (P = 0,04) hoër was as in individue sonder MIV infeksie. Daar was verhoogde oksidatiewe stres soos bewys deur verhoogde katalase aktiwiteit, malondialdehied (MDA) en gekonjugeerde diëne (CDs), saam met 'n ooreenstemmende afname in anti-oksidant kapasiteit in MIV-positiewe individue met laer CD4-tellings. NAC in kombinasie met vitamien C, het die aktivering van CD4 T-selle beduidend verminder (P = 0,0018), 'n effek wat groter was in vergelyking met elke antioksidant alleen. NAC en vitamien C alleen, en in kombinasie het beduidend die uitdrukking van die merkers van apoptose, Annexin V en 7-AAD verminder (P = 0,05, P = 0.012 en P<0,0001). Wat belangrik is, is dat die afname in MDA waardes as gevolg van antioksidante in kombinasie, 'n beduidende styging in die glutatioon redoks verhouding in die MIV-positiewe groep tot gevolg gehad het. Hierdie resultate het aangetoon dat die respiratoriese uitbarsting en LPS-geïnduseerde aktivering belangrike bydraende faktore mag wees in inflammasie-verwante oksidatiewe stres in MIV-infeksie, wat kan bydra tot die uitputting van CD4 T-selle in die asimptomatiese stadium van MIV-infeksie. Hierdie resultate dui ook aan dat die moontlike inhiberende effekte van NAC en vitamien C in kombinasie, immuun aktivering en geïnduseerde oksidatiewe stres kan beperk. Van belang is die feit dat hierdie studie bewys het dat in die asimptomatiese stadium van MIV-infeksie, CD4 T-selle weens stimulasie dieselfde gereageer het as dié van mense sonder MIV infeksie. Dit het aangedui dat antioksidant verdediging in hierdie stadium nog funksioneel was, en dat 'n toepaslike vroeë intervensie op hierdie stadium beskermend teen immuun-sel oksidatiewe skade kan wees. Tot die beste van ons kennis, is hierdie studie die eerste om 'n geïntegreerde benadering te gebruik, waar plasma vlakke van antioksidant status saam met RBC antioksidant ensiemaktiwiteit, oksidatiewe skade (lipied peroksiidasie), inflammasie, sellulêre vlakke van immuunaktivering, en potensiële beskermende ingrypings ondersoek is in die evaluering van die probleem van oksidatiewe stres in MIV-infeksie wat tot inflammasie lei. Gebaseer op dié resultate, word dit in die vooruitsig gestel dat 'n insig in die status van immuunaktivering, inflammasie, en oksidatiewe stress van pasiënte, dit moontlik sal maak vir langtermyn- beplanning om vir elke pasiënt individuele terapie voor te skryf. Hierdie benadering kan 'n direkte impak op die sorg van pasiënte in hulpbron-beperkte gebiede soos sub-Sahara Afrika hê.

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